Animal and plant cells have been cultivated by various cultivation techniques. The technique of cell cultivation is one of the basic techniques used in various biological fields. In the fields of life sciences, this technique of cell cultivation is particularly indispensable in developing drugs and elucidating the pathological mechanism. In recent years, cultivation techniques have been developed not only for research purpose but also for use in commercial scale cultivation of the cells in the biology, medicine, immunology, and other fields. For example, in the field of medicine, tissue cells are cultivated for use in prosthetics such as artificial organ, artificial mandible, and artificial skin.
In such cell cultivation, the cells are cultivated in a certain container with a culture medium containing nutrient components. The cell cultures are divided by its nature into two major categories, namely, the cell culture floating in the culture medium and the cell culture attached to the bottom surface of the container. Many animal cells are anchorage dependent, and they only grow when the they are attached to a substrate, and they are incapable of surviving for a long time when they are flowing in the culture medium. Accordingly, a substrate for adhesion of the cells is required in cultivating such anchorage dependent cells.
Examples of typical cell culture substrate used in laboratories include dish, flask, and multi plate, and commercially available such substrates include those prepared by providing a surface of a polystyrene molded article with hydrophilicity by low temperature plasma treatment, corona discharge treatment, and the like. These devices are widely used in the cultivation of anchorage dependent cells including fibroblast, smooth muscle cell, endothelial cell, and cornea cell for both established cells and initial cells. These devices are also widely used for non-anchorage dependent floating cells such as lymphocytes which have been established as a blood cell line.
However, some cells exhibit insufficient growth or unfavorable growth morphology even if such cell propagated on these cell culture devices, and this situation is significant in the case of initial culture. In view of such situation, the cultivation surface of the container has often been coated with an extracellular matrix such as collagen, gelatin or a adhesion factor such as fibronectin, laminin, and vitronectin to thereby improve adhesion and propagation ability of the cells.
For example, non-patent documents 1, 2, 3, and 4 and patent documents 1, 2, and 3 as described below disclose production of cultivated epithelium and epidermis by using a dish coated with collagen, a collagen gel, a collagen sponge, a collagen sheet having a three dimensional structure produced by molecular crosslinking, a collagen sponge formed with through holes, and the like for the cell culture substrate, inoculating a cell such as human fibroblast or human keratinocyte on such substrate, and cultivating the cell; and producing cultivated mucosa and skin by forming a layer of human keratinocyte on the human fibroblast.
The collagen used in forming the collagen coating layer is Type I collagen from connective tissue of an animal which has been solubilized by using an acid or enzyme. The collagen coated layer can be produced by coating this collagen on a culture dish or the like followed by drying. A typical example of the collagen which can serve such cell culture substrate is the one solubilized and extracted from bovine or porcine connective tissue. However, use of such bovine or porcine collagen has become increasingly difficult in view of the problems such as BSE (bovine spongiform encephalopathy) and foot and mouth disease. In addition, there is a need for a cell cultivation method which has an improved cell propagation efficiency than the method using such conventional cell culture substrate.
In cultivating a neural cell, a surface having coated thereon a polylysine such as poly-D-lysine and poly-L-lysine is often used. The polylysine coating facilitates adhesion of the neural cell, and neural cell lines cultivated by using such coating exhibit good propagation morphology with sufficient extension of the neurite.
As described above, the polylysine has various favorable properties for the cultivation of neural cells. The polylysine, however, has a drawback of insufficient stability. When a polylysine is coated on the cultivation device commonly used in the art as described above, the activity of polylysine is lost in 2 weeks when stored at room temperature, and in 1 month when stored at 4° C. In addition, the coated device can not be sterilized because of such insufficient stability. Accordingly, use of a cultivation device coated with the polylysine requires an inconvenient step of coating of the polylysine in a sterile environment on a preliminarily sterilized cultivation device before its use, and this is a quite complicated process, and the thus prepared device could be stored only for about 1 month even if stored in the refrigerator.
As described above, coating of the polylysine on the cultivation device commonly used in the art takes considerable amount of work, and one reason has been the instability of the polylysine after its coating. In addition, this coating required use of a sterile environment such as use of a clean bench. Accordingly, selling of a cell cultivation device having the polylysine preliminarily coated thereon would need a number of steps carried out in sterile environment as well as storage under controlled environment that would result increase in the cost.    Non-Patent Document 1: Rinsho Kagaku (The Journal of Clinical Science), vol. 34, No. 9, Y. Shirakata and K. Hashimoto, “Regenerative medicine X: regeneration mechanism of skin-epidermis and burn treatment using cultivated epidermis sheet”, pages 1283 to 1290    Non-Patent Document 2: Nagoya University Press, issued on 1999/10/10, “Fundamentals and application of textile engineering: Tissue engineering” ed. by M. Ueda, K. Matsuzaki and N. Kumagaya, Cultured skin, pages 107 to 117    Non-Patent Document 3: M. Ueda and K. Hata, Skin and Mucosa, Pharma Media Vol. 18, No. 1, 2000, pages 25 to 29, 2000    Non-Patent Document 4: Nagoya University Press, issued on 1999/10/10, “Basic and applied textile engineering: Tissue engineering” ed. by M. Ueda, M. Ueda and K. Hata, oral mucosa, pages 118 to 127    Patent Document 1: Japanese Patent Application Laid-Open No. 6-292568    Patent Document 2: Japanese Patent Application Laid-Open No. 8-243156    Patent Document 3: Japanese Patent Application Laid-Open No. 9-47503